Infections occurring among cultured fish, principally salmonid fish, are a major source of economic loss in the fish rearing industry all over the world. It has hitherto been attempted to reduce or eliminate losses resulting from certain infections of bacterial etiology (e.g. furunculosis caused by Aeromonas salmonicida, enteric redmouth caused by Yersinia ruckerii and vibriosis caused by Vibrio anguillarum) by means of chemotherapeutic agents such as sulfa drugs or oxytetracycline. The use of antibacterial agents, however, is rather expensive and, furthermore, drug resistant strains of the various pathogenic bacteria have been known to develop. Besides, the use of antibacterial agents offers no protection against diseases of a viral etiology.
Efforts have therefore been made to develop a vaccine against selected fish pathogens. Thus, a vaccine has been developed against Y. ruckerii (Tebbit et al. in: Developments in Biological Standardization, Vol. 49, International Symposium on Fish Biologics: Serodiagnostics and Vaccines, W. Heunessen and D. P. Anderson (eds.), 1981, pp. 395-402), and V. anguillarum (Amend and Johnson in: Developments in Biological Standardization, Vol. 49, International Symposium on Fish Biologics: Serodiagnostics and Vaccines, W. Heunessen and D. P. Anderson (eds.), 1981, pp. 403-418; Agius et al., J. Fish Dis. 6, 1983, pp. 129-134). These vaccines are based on formalin-killed virulent bacteria. The efficacy of these vaccines has been tested and it has been shown that the route of administration of the vaccines plays an important part for the strength of the resulting immune response (Kawano et al., Bull. Jpn. Soc. Sci. Fish. 50, 1984, pp. 771-774; Ward et al., in Fish Immunology, M. J. Manning and M. F. Tatner (eds.), 1985, pp. 221-229). Experiments have shown that injection of a vaccine preparation gives by far the best result with the longest duration of the immunization, while immersion and oral administration give a less efficient protection against the infections in question.
Further, a vaccine comprising chloroform-inactivated whole cells, soluble antigen and combined whole cell and soluble antigen of an avirulent strain of Aeromonas salmonicida has been shown to protect fish against furunculosis (Cipriano et al., J. World Maricul. Soc., 1983, 14, 201-211).
An improved understanding of the pathogenic properties of the fish pathogens against which it is desired to develop a useful immune response is important for the construction of an efficient vaccine. In spite of the widespread occurrence of epizootics in fish farms caused by a variety of different pathogens, both bacterial and viral, studies of the properties contributing to virulence have so far been limited in number. In the case of V. anguillarum, a number of studies have established the existence in this bacterium of a virulence plasmid of 65 kb (kilobase pairs) which encodes a complex system important to the ability of the bacterium to take up Fe.sup.3+ (Crosa, Nature 284, 1980, pp. 566-568). It has been shown for many pathogenic microorganisms that the ability to take up Fe.sup.3+ is important for the bacteria during the process of infection. If V. anguillarum is cured of the virulence plasmid, it becomes avirulent for fish (Crosa et al., Infect. Immun. 27, 1980, pp. 897-902).
Furthermore, a number of publications describe possible virulence determinants such as serum resistance (Trust et al., Infect. Immun. 34, 1981, pp. 702-707), hemolysin (Munn, FEMS Micribiol. Lett. 3, 1978, pp. 265-268), extracellular protease (Inamura et al., Bull. Jap. Soc. Sci. Fish. 51, 1985, pp. 1915-1920) and other extracellular factors (Kodama et al., Am. J. Vet. Res. 45, 1984, pp. 2203-2207). It has been possible in one way or another to correlate these virulence determinants to the outbreak of disease. However, it has not yet been conclusively proved that these bacterial products are virulence determinants which are important for the pathogenicity of the bacterium.
In recent years, interest has increased in developing live vaccines based on live attenuated bacterial strains for use in the prevention of human diseases, in particular against enteric agents (Hone et al., J. Inf. Dis. 156, 1987, pp. 167-174; Wahdan et al., J. Inf. Dis. 145, 1982, pp. 292-295). Cipriano and Stasliper, Prog. Fish-Cult., 44, 1982, 167-169, have disclosed a vaccine comprising an attenuated A. salmonicida strain. Live vaccines generally have the advantage over vaccines based on killed pathogens or bacterial components that they confer a higher degree of immunity as well as a more prolonged effect, and is more complete than when single components such as antigens are administered. Furthermore, they may require a smaller efficacious dosage than killed pathogens or single components. Live vaccines may also be less expensive to produce than those based on a purified single component, no purification step being required. It would therefore be an advantage to develop a live vaccine for administration to fish requiring a lower immunization dosage than existing fish vaccines based on killed pathogens or membrane components.
A need clearly exists to develop a vaccine against fish pathogens which can provoke a long-lasting and efficient immunological protection against a broad spectrum of fish pathogens after immersion of the fish into the vaccine. Ideally, such a vaccine should comprise a bacterial strain which possesses as many characteristics of the pathogenic microorganisms as possible, but without their ability to cause disease. It is of special significance to select vaccine strains which can penetrate into and proliferate in the fish body, thereby eliciting a strong immune response.